CN114645076A - miR-589-3p horizontal in-situ hybridization detection kit and detection method - Google Patents

miR-589-3p horizontal in-situ hybridization detection kit and detection method Download PDF

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CN114645076A
CN114645076A CN202210192180.2A CN202210192180A CN114645076A CN 114645076 A CN114645076 A CN 114645076A CN 202210192180 A CN202210192180 A CN 202210192180A CN 114645076 A CN114645076 A CN 114645076A
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situ hybridization
probe
fluorescence
slide
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CN114645076B (en
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廖兴华
沈超
李佳蓬
项园
戴周彤
张慧敏
王君
黄优
宗启贝
李乐威
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Wuhan University of Science and Engineering WUSE
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Abstract

The invention provides a miR-589-3p horizontal in-situ hybridization detection kit and a detection method, wherein the kit comprises a CD30 monoclonal antibody, a miR-589-3p probe, a confining liquid, a stationary liquid, a hybridization liquid and a counterstain. The invention adopts the CD30 immunofluorescence antibody and the miR-589-3p fluorescence in situ hybridization probe to detect the pathological change condition of the cancer, and overcomes the technical problems of false positive and poor penetration in the fluorescence in situ hybridization detection.

Description

miR-589-3p horizontal in-situ hybridization detection kit and detection method
Technical Field
The invention relates to the technical field of medicines, in particular to a miR-589-3p horizontal in-situ hybridization detection kit and a detection method.
Background
Fluorescence In Situ Hybridization (FISH) is an emerging molecular cytogenetic technology, and is a non-radioactive in situ hybridization technology developed on the basis of the original radioactive in situ hybridization technology at the end of the 80 th 20 th century. The technology is widely applied to the fields of animal and plant genome structure research, chromosome fine structure variation analysis, virus infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research.
The basic principle of FISH is that known labeled single-stranded nucleic acid is used as a probe, and the known labeled single-stranded nucleic acid is combined with unknown single-stranded nucleic acid in a material to be detected in an anisotropic manner according to the base complementary principle to form hybrid double-stranded nucleic acid which can be detected. Since the DNA molecules are linearly arranged on the chromosome along the longitudinal axis of the chromosome, the probe can directly hybridize with the chromosome to locate a specific gene on the chromosome. Compared with the traditional radioactive labeling in situ hybridization, the fluorescence in situ hybridization has the characteristics of rapidness, strong detection signal, high hybridization specificity, multiple staining and the like, so that the fluorescence in situ hybridization is generally concerned in the field of molecular cytogenetics.
Micro RNA (miRNA) is an endogenous small non-coding RNA with a length of about 18-25 nucleotides, and it usually inhibits its translation or accelerates its degradation by binding to the 3 'untranslated region (3' UTR) of target messenger RNA (mrnas) to regulate mRNA expression, thereby playing an important role in the aspects of organism development, disease development, etc., and its role in tumors is also more and more emphasized by people. The research related to miR-589 mainly aims at the research of miR-589-5P, and the research of miR-589-5P adopts high-throughput gene chip analysis technologies such as Northern hybridization, an expression chip, real-time fluorescence quantitative PCR and Solexa sequencing, such as: the expression level of miR-589-5P is in negative correlation in liver cancer tissues, and miR-589-5P can be used for diagnosing acute gout. There is no research on the application of miR-589-3P level in-situ hybridization in the detection of precancerous lesion. Meanwhile, the miRNA fluorescence in-situ hybridization widely used at present has the technical problem of false positive caused by poor autofluorescence penetrability, and the detection sensitivity is reduced.
Disclosure of Invention
In view of the above, the invention provides the miR-589-3p level in-situ hybridization detection kit and the detection method, which can reduce false positive and improve detection sensitivity.
The technical scheme of the invention is realized as follows: the invention provides a miR-589-3p horizontal in-situ hybridization detection kit, which is characterized in that: comprises CD30 monoclonal antibody, miR-589-3p probe, hybridization solution, stationary liquid, confining liquid and counterstain.
On the basis of the technical scheme, preferably, the CD30 monoclonal antibody is a fluorescence-labeled CD30 monoclonal antibody, the miR-589-3p probe is a miR-589-3p fluorescence in situ hybridization probe, and the nucleotide sequence of the miR-589-3p fluorescence in situ hybridization probe is shown as SEQ ID NO: 1.
On the basis of the technical scheme, preferably, the hybridization solution comprises 15-30% of dextran sulfate by mass fraction, 2-5mol/L of formamide by concentration, 1-2mol/L of biological guanidine isothiocyanate by concentration, 6 XSSC and 1-3% of Triton-100 by volume fraction, and the solvent is deionized water.
On the basis of the technical scheme, preferably, the fixing solution comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
On the basis of the above technical scheme, preferably, the blocking solution comprises 1-3% by mass of BSA, 3-5% by mass of Tween20 and 10 × SSC, and the solvent is deionized water.
On the basis of the technical scheme, preferably, the counterstain is PI/anti-dye or DAPI/anti-dye liquor.
The invention also provides a use method of the miR-589-3p level in situ hybridization detection kit, which comprises the following steps:
s1, preparing tablets: the cell sample is harvested and transferred into a centrifuge tube, the supernatant is discarded after centrifugation, then the cell suspension is prepared through operations of hypotonic, pre-fixing and fixing, the cell suspension is absorbed and dropped onto a glass slide, and the cell slide is obtained after aging;
s2, pre-hybridization treatment: sealing, perforating and dehydrating the cell slide to obtain a dry slide;
s3, denaturation treatment: the miR-589-3p fluorescence in situ hybridization probe and the cell slide are subjected to denaturation treatment at the temperature of 70-80 ℃;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, dripping the mixture to a denatured cell slide hybridization area, covering a cover glass, sealing the edge, and performing hybridization incubation;
s5, washing: after incubation is finished, the slide is eluted for many times, soaked in deionized water and then naturally dried;
s6, sealing and observing: and (4) dripping the counterstain on the dried glass slide, and observing the glass slide under a fluorescence microscope.
Based on the above technical solution, preferably, the denaturation treatment in step S3 is: immersing the cell slide in 70-80 ℃ denaturing solution with 70% of formamide/2 XSSC volume fraction for denaturation for 2-3 min; and (3) incubating the miR-589-3p fluorescence in-situ hybridization probe in a constant-temperature water bath at 70-80 ℃ for 5min, and then, incubating the probe at 0 ℃ for 5-10 min to denature the double-stranded DNA probe.
Compared with the prior art, the miR-589-3p horizontal in-situ hybridization detection kit and the detection method have the following beneficial effects: the invention adopts the CD30 immunofluorescence antibody and the miR-589-3p fluorescence in situ hybridization probe to detect the pathological change condition of the cancer, and overcomes the technical problems of false positive and poor penetration in the fluorescence in situ hybridization detection. The kit can rapidly complete hybridization within 2 hours, and greatly shortens the hybridization time compared with 16-24 hours of the conventional probe. The kit can detect the expression quantity of the miR-589-3p gene on the mRNA level, and realizes the screening of the mRNA level at the early stage of various cancer lesions.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a positive microscopic examination chart of lung cancer detected by in situ hybridization according to an example;
FIG. 2 is a positive microscopic examination chart of in situ hybridization detection of breast cancer in the second embodiment;
FIG. 3 is a positive microscopic examination image of the three in-situ hybridization assay for gastric cancer in the example;
FIG. 4 is a positive microscopic examination image of in situ hybridization assay for gastric cancer in comparison example.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention firstly provides a miR-589-3p horizontal in-situ hybridization detection kit, which comprises a CD30 monoclonal antibody, a miR-589-3p probe, confining liquid, fixing liquid, hybridization liquid, eluent and counterstain.
The CD30 monoclonal antibody is CD30 monoclonal antibody marked by fluorescence; the miR-589-3p probe is a miR-589-3p fluorescence in situ hybridization probe, and the nucleotide sequence of the probe is shown in SEQ ID NO: 1.
The hybridization solution comprises 15-30% of dextran sulfate by mass fraction, 2-5mol/L formamide by mass fraction, 1-2mol/L biological guanidine isothiocyanate by mass fraction, 6 XSSC and 1-3% of Triton-100 by volume fraction, and the solvent is deionized water.
The fixing solution comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
The perforating agent comprises 0.5 percent of Triton X-100 by mass and 3 percent of BSA by mass.
The blocking liquid comprises BSA with the mass fraction of 1-3%, Tween20 with the mass fraction of 3-5% and 10 XSSC, and the solvent is deionized water.
The counterstain is PI/anti-fiade or DAPI/anti-fiade dye solution.
Example one
The application method of the miR-589-3p level in-situ hybridization detection kit takes a lung cancer cell sample as a detection sample. The method comprises the following steps:
s1, preparing tablets: sucking the captured lung cancer cell sample to a sharp-bottomed centrifuge tube, centrifuging, rotating at 2500 rpm for 10min, and removing a supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, the mixture is blown, beaten and uniformly mixed, and then the mixture is placed in a 37 ℃ incubator for 30 min; adding 2mL of stationary liquid, blowing and beating uniformly, and centrifuging at 2500 rpm for 10 min; absorbing the supernatant, adding 5mL of stationary liquid, blowing, uniformly mixing, fixing for 10min, centrifuging at 2500 rpm for 10min, and repeating the steps until the cell sediment is washed clean; then sucking the supernatant, adding a proper amount of stationary liquid to prepare cell suspension with proper concentration; then 5 mul of cell suspension is absorbed and dripped on a glass slide, and the lung cancer cell slide is obtained after 1 hour of aging at 60 ℃; the stationary liquid is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 2: 1.
S2, slide treatment before hybridization: adding 100ul of blocking solution into the lung cancer cell slide, blocking at room temperature for 10min, and rinsing with 2 XSSC (pH 7.0) for 5 min; then 100ul of perforating agent is dripped, and after the sealing is carried out for 10min at the room temperature, 2 XSSC (pH 7.0) is rinsed for 5 min; then placing the glass slide in 70Vt percent ethanol, 85Vt percent ethanol and 100Vt percent ethanol in sequence, dehydrating for 2min respectively, and naturally drying the glass slide; the blocking solution comprises BSA with the mass fraction of 1%, Tween20 with the mass fraction of 3% and 10 XSSC, and the solvent is deionized water. The perforating agent comprises 0.2 percent of Triton X-100 by mass and 1 percent of BSA by mass.
S3, denaturation treatment: immersing the lung cancer cell slide in 70 ℃ denaturing solution with 70% of formamide/2 XSSC volume fraction for denaturation for 2 min; incubating the miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 70 ℃ for 5min, and then placing the probe at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in situ hybridization probe and hybridization solution, dripping 10 mu L of the mixture into a cell drop hybridization area, immediately covering a cover glass, uniformly spreading the probe without bubbles under the cover glass, sealing the edge with resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises 15% of dextran sulfate by mass fraction, 2mol/L of formamide by volume fraction, 1mol/L of biological guanidine isothiocyanate by volume fraction, 6 XSSC and 1% of Triton-100 by volume fraction, and the solvent is deionized water
S5, washing: taking the slide specimen out of the incubator, and slightly uncovering the cover glass by a blade; then washing in preheated 40 deg.C formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5 min; then washing in 1 XSSC preheated to 40 ℃ for 3 times, each time for 5 min; the slide specimen was washed in deionized water at room temperature. And taking out the slide and naturally drying.
S6, sealing and observing: 200 μ L of the counterstain solution PI/antipade was dropped on the slide specimen, covered with a cover slip, and the slide was observed under a fluorescence microscope, and the result is shown in FIG. 1.
Example two
The application method of the miR-589-3p level in-situ hybridization detection kit takes a breast cancer cell sample as a detection sample. The method comprises the following steps:
s1, preparing tablets: sucking the captured mammary gland cell sample to a pointed-bottom centrifuge tube, centrifuging at 2500 rpm for 10min, and removing supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, the mixture is blown, beaten and uniformly mixed, and then the mixture is placed in a 37 ℃ incubator for 30 min; adding 2mL of stationary liquid, blowing, beating and mixing uniformly, and centrifuging at 2500 rpm for 10 min; absorbing the supernatant, adding 5mL of stationary liquid, blowing, uniformly mixing, fixing for 10min, centrifuging at 2500 rpm for 10min, and repeating the steps until the cell sediment is washed clean; then sucking the supernatant, adding a proper amount of stationary liquid to prepare cell suspension with proper concentration; then 5 mu L of cell suspension is absorbed and dripped on a glass slide, and the cell suspension is aged for 1 hour at the temperature of 60 ℃ to obtain a breast cancer cell slide; the stationary liquid is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 5: 1.
S2, slide treatment before hybridization: adding 100ul of blocking solution into the breast cancer cell slide, blocking at room temperature for 10min, and rinsing with 2 XSSC (pH 7.0) for 5 min; then 100ul of perforating agent is dripped, and after the sealing at room temperature for 10min, 2 XSSC (pH 7.0) is used for rinsing for 5 min; then, placing the glass slide in 70Vt percent ethanol, 85Vt percent ethanol and 100Vt percent ethanol in sequence, dewatering for 2min respectively, and then naturally drying the glass slide; the blocking liquid comprises BSA (bovine serum albumin) with the mass fraction of 3%, Tween20 with the mass fraction of 5% and 10 XSSC, and the solvent is deionized water. The perforating agent comprises 0.5 percent of Triton X-100 by mass and 3 percent of BSA by mass.
S3, denaturation treatment: immersing the breast cancer cell slide in a denaturing solution with a volume fraction of 70% formamide/2 XSSC at 80 ℃ for denaturation for 2 min; incubating the miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 80 ℃ for 5min, and then placing the probe at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in situ hybridization probe and hybridization solution, dripping 10 mu L of the mixture into a cell drop hybridization area, immediately covering a cover glass, uniformly spreading the probe without bubbles under the cover glass, sealing the edge with resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises 30% of dextran sulfate by mass fraction, 5mol/L of formamide by volume fraction, 2mol/L of biological guanidine isothiocyanate by volume fraction, 6 XSSC and 3% of Triton-100 by volume fraction, and the solvent is deionized water
S5, washing: taking the slide specimen out of the incubator, and slightly uncovering the cover glass by a blade; then washing in preheated 40 deg.C formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5 min; then washing in 1 XSSC preheated to 40 ℃ for 3 times, each time for 5 min; the slide specimen was washed in deionized water at room temperature. And taking out the slide and naturally drying.
S6, sealing and observing: 200 μ L of counterstain DAPI/antistade solution was dropped on the slide specimen, covered with a cover slip, and the slide was observed under a fluorescence microscope, and the result is shown in FIG. 2.
EXAMPLE III
An application method of the miR-589-3p level in-situ hybridization detection kit takes a gastric cancer cell sample as a detection specimen. The method comprises the following steps:
s1, preparing tablets: sucking the captured gastric cancer cell sample to a sharp-bottomed centrifuge tube, centrifuging at 2500 rpm for 10min, and removing supernatant; then 8mL of 0.075mol/L KCL solution at 37 ℃ is added, the mixture is blown, beaten and uniformly mixed, and then the mixture is placed in a 37 ℃ incubator for 30 min; adding 2mL of stationary liquid, blowing, beating and mixing uniformly, and centrifuging at 2500 rpm for 10 min; sucking off the supernatant, adding 5mL of stationary liquid, blowing, uniformly mixing, fixing for 10min, centrifuging at 2500 rpm for 10min, and repeating the steps until the cell sediment is washed clean; then sucking the supernatant, adding a proper amount of stationary liquid to prepare cell suspension with proper concentration; then 5 mul of cell suspension is absorbed and dropped on a glass slide, and the cell suspension is aged for 1 hour at the temperature of 60 ℃ to obtain a gastric cancer cell slide; the stationary liquid is obtained by mixing methanol and glacial acetic acid according to the volume ratio of 3: 1.
S2, pre-hybridization slide treatment: adding 100ul of blocking solution into the gastric cancer cell slide, blocking at room temperature for 10min, and rinsing with 2 XSSC (pH 7.0) for 5 min; then 100ul of perforating agent is dripped, and after the sealing at room temperature for 10min, 2 XSSC (pH 7.0) is used for rinsing for 5 min; then placing the glass slide in 70Vt percent ethanol, 85Vt percent ethanol and 100Vt percent ethanol in sequence, dehydrating for 2min respectively, and naturally drying the glass slide; the blocking solution comprises BSA with the mass fraction of 2%, Tween20 with the mass fraction of 4% and 10 XSSC, and the solvent is deionized water. The perforating agent comprises 0.3 percent of Triton X-100 by mass and 2 percent of BSA by mass.
S3, denaturation treatment: immersing the gastric cancer cell slide in a denaturing solution with the volume fraction of 70% formamide/2 XSSC at 80 ℃ for denaturation for 2 min; incubating the miR-589-3p fluorescent in-situ hybridization probe in a constant-temperature water bath at 80 ℃ for 5min, and then placing the probe at 0 ℃ for 5min to denature the double-stranded DNA probe;
s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in situ hybridization probe and hybridization solution, dripping 10 mu L of the mixture into a cell drop hybridization area, immediately covering a cover glass, uniformly spreading the probe without bubbles under the cover glass, sealing the edge with resin, placing the glass on a hybridization instrument, denaturing at 75 ℃ for 2 minutes, and incubating at 37 ℃ for 2 hours; the hybridization solution comprises dextran sulfate with the mass fraction of 20%, formamide with the concentration of 4mol/L, biological guanidine isothiocyanate with the concentration of 1.5mol/L, 6 XSSC, Triton-100 with the volume fraction of 2%, and deionized water as a solvent;
s5, washing: taking the slide specimen out of the incubator, and slightly uncovering the cover glass by a blade; then washing in preheated 40 deg.C formamide with volume fraction of 50% and 2 XSSC for 3 times, each time for 5 min; then washing in 1 XSSC preheated to 40 ℃ for 3 times, each time for 5 min; the slide specimen was washed in deionized water at room temperature. And taking out the slide and naturally drying.
S6, sealing and observing: dripping 200 mu L of counterstain solution DAPI/anti-formaldehyde on a slide specimen, covering a cover glass, and observing the slide under a fluorescence microscope; the results are shown in FIG. 3.
In the comparative example, the existing fluorescence in situ hybridization method is taken as a first comparative example, no antibody exists, only the probe is hybridized with the cells, the number of detected cases in each example is 80, wherein 50 positive samples are obtained, the detection result of the first comparative example is the same as that of the first example, and the detection result is as follows:
TABLE 1 cancer test results
Figure BDA0003525297010000091
Figure BDA0003525297010000101
As shown in Table 1, the CD30 immunofluorescence antibody and the miR-589-3p fluorescence in situ hybridization probe are adopted to detect cancer lesions, the technical problems of false positive and poor penetration in fluorescence in situ hybridization detection are solved, the kit can rapidly complete hybridization within 2 hours, and the detection time is greatly shortened.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan science and technology university
<120> miR-589-3p horizontal in situ hybridization detection kit and detection method
<141> 2022-02-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 106
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccgttcagaa caaatgccgg ctgttaatgc taatcgtgat aggggttttt gcctccaact 60
gactcctaca tattagcatt aacagagtgc agggtccgag gtattc 106

Claims (8)

  1. The kit for detecting the horizontal in-situ hybridization of miR-589-3p is characterized by comprising the following components in parts by weight: comprises CD30 monoclonal antibody, miR-589-3p probe, hybridization solution, stationary liquid, confining liquid and counterstain.
  2. 2. The miR-589-3p level in situ hybridization detection kit of claim 1, which is characterized in that: the CD30 monoclonal antibody is a fluorescence labeled CD30 monoclonal antibody, the miR-589-3p probe is a miR-589-3p fluorescence in situ hybridization probe, and the nucleotide sequence of the miR-589-3p fluorescence in situ hybridization probe is shown in SEQ ID NO 1.
  3. 3. The miR-589-3p level in situ hybridization detection kit of claim 1, which is characterized in that: the hybridization solution comprises 15-30% of dextran sulfate by mass fraction, 2-5mol/L of formamide by mass fraction, 1-2mol/L of biological guanidine isothiocyanate by mass fraction, 6 XSSC and 1-3% of Triton-100 by volume fraction, and the solvent is deionized water.
  4. 4. The miR-589-3p level in situ hybridization detection kit of claim 1, which is characterized in that: the fixing solution comprises methanol and glacial acetic acid, and the volume ratio of the methanol to the glacial acetic acid is (2-5): 1.
  5. 5. The miR-589-3p level in situ hybridization detection kit of claim 1, which is characterized in that: the blocking liquid comprises BSA (bovine serum albumin) with the mass fraction of 1-3%, Tween20 and 10 XSSC with the mass fraction of 3-5%, and a solvent is deionized water.
  6. 6. The miR-589-3p level in situ hybridization detection kit of claim 1, which is characterized in that: the counterstain is PI/anti-fiade or DAPI/anti-fiade staining solution.
  7. 7. The use method of the miR-589-3p level in situ hybridization detection kit of any one of claims 1-6, which is characterized in that: the method comprises the following steps:
    s1, preparing tablets: the cell sample is harvested and transferred into a centrifuge tube, the supernatant is discarded after centrifugation, then the cell suspension is prepared through operations of hypotonic, pre-fixing and fixing, the cell suspension is absorbed and dropped onto a glass slide, and the cell slide is obtained after aging;
    s2, pre-hybridization treatment: sealing, perforating and dehydrating the cell slide to obtain a dry slide;
    s3, denaturation treatment: the miR-589-3p fluorescence in situ hybridization probe and the cell slide are denatured at 70-80 ℃;
    s4, hybridization incubation: uniformly mixing a fluorescence-labeled CD30 monoclonal antibody, a denatured miR-589-3p fluorescence in-situ hybridization probe and hybridization solution, dripping the mixture to a denatured cell slide hybridization area, covering a cover glass, sealing the edge, and performing hybridization incubation;
    s5, washing: after incubation is finished, the cell slide is eluted for many times, soaked in deionized water and then naturally dried;
    s6, sealing and observing: the counterstain was dropped onto the dried cell slide and the slide was observed under a fluorescence microscope.
  8. 8. The use method of the miR-589-3p level in situ hybridization detection kit of claim 7, which is characterized in that: the denaturation treatment in step S3 is: immersing the cell slide into 70-80 ℃ denaturing solution with 70% of formamide/2 XSSC in volume fraction for denaturation for 2-3 min; and (3) incubating the miR-589-3p fluorescence in-situ hybridization probe in a constant-temperature water bath at 70-80 ℃ for 5min, and then placing the probe at 0 ℃ for 5-10 min to denature the double-stranded DNA probe.
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